Glycosylation is a common post-translational modification having significant effects on protein characteristics like solubility, folding and receptor binding activity [1-3]. An increasing number of diseases have shown association with changes in glycosylation, including various forms of cancer, autoimmunity and congenital disorders [4-8]. Because of this, the profiling of glycans of individual proteins or more complex samples like plasma can serve as an important clinical biomarker [9]. In addition, glycans are known to affect the activity, stability and immunogenicity of biopharmaceuticals, requiring careful monitoring and control [10, 11].
An important characteristic of glycosylation is the presence of sialic acids (such as N-acetylneuraminic acid and N-glycolylneuraminic acid) [12]. These monosaccharides have shown many roles in cellular communication, interact with specific types of lectins, and have been associated with cancer and metastaticity [13-15]. In case of human glycosylation, sialic acids may be attached to a terminal galactose either by α2,6 or α2,3 glycosidic linkage, showing different functionality as a consequence. α2,3-linked N-acetylneuraminic acids for example, are specifically required for formation of sialyl Lewis X structures, which have shown to be indicative for metastasis of several types of cancer [16-19], whereas α2,6-linked sialic acids show roles in galectin inhibition, thereby promoting cell survival [20-22]. With an increasing need for analysis, the development of methods for high-throughput (HTP) glycomics is of great interest.
A technique well suited for glycomic studies is matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS), as it can rapidly provide information on the composition, sequence and branching of glycan structures [23]. Sialic acids, however, are attached to glycans by a relatively weak bond, often leading to loss of the residue by in-source and metastable decay during the respective ionization and acceleration phases in the mass spectrometer. In addition, sialylated glycan species tend to show high variability in salt adduction, resulting in multiple signals for single glycan compositions. Moreover, a carboxyl group such as present on a sialic acid facilitates negative ionization by MALDI considerably, generating a bias in signal intensity when comparing acidic and neutral oligosaccharides, preventing relative quantification of mixed samples [24].
One way to improve MALDI-TOF-MS measurement of sialylated glycans is by derivatisation [25, 26]. In particular, chemical modifications of the sialic acid carboxyl group can prevent metastable decay to a large degree, and the resulting reaction product can be analyzed in positive-mode MALDI-TOF-MS together with the non-sialylated species [24, 25, 27]. Interestingly, reactions conditions have been developed which allow derivatisation of sialic acids in a linkage-dependent manner, differentially derivatising α2,3-linked sialic acids (prone to lactone formation) and α2,6-linked sialic acids (undergoing other chemical modification such as esterification and amidation). Methods described in literature involve methyl esterification or (methyl)amidation, but these typically require highly purified glycan samples, harsh conditions or long reaction times, and do not show complete linkage-specificity [28-30]. While these procedures are informative for the analysis of sialylated oligosaccharides, suitability for HTP analysis of complex samples is limited.
It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.